Hepatocellular carcinoma protein marker, and method for detection of hepatocellular carcinoma using the same

ABSTRACT

A method of detecting hepatocellular carcinoma includes using an isolated protein including an amino acid sequence represented by SEQ ID NO: 1.

CROSS REFERENCE TO RELATED APPLICATIONS

The present Application is a Divisional Application of U.S. Pat. No.12/452,216, filed on Dec. 22, 2009, which is pending, the contents ofwhich are hereby incorporated by reference.

REFERENCE TO SEQUENCE LISTING

Incorporated by reference herein is the sequence Listing including inthe text file having a file name “SequenceListing.txt” which was createdon May 15, 2012 and has a size of 7 KB.

TECHNICAL FIELD

This invention relates to a phosphorylating-modified, i.e.phosphorylated, protein marker that can be used in detection ofhepatocellular carcinoma and a method of detecting a hepatocellularcarcinoma including a step of using the protein marker.

BACKGROUND ART

The hepatocellular carcinoma is one of epithelial malignant tumorsdeveloped as a primary carcinoma in a liver and formed of tumor cellssimilar to hepatocytes. In general, a large number of tumors are formedin a liver and are likely to grow and progress in a hepatic vessel, anda tumor thrombosis is frequently formed in a portal vein. Thehepatocellular carcinoma often breaks out in the Asian region includingJapan and the African region, and cirrhosis concurs in many cases.

As a marker for detecting hepatocellular carcinoma, use is made havebeen conventionally of α-fetoprotein (AFP) and PIVKA-II (see Non-patentDocument 1), KM-2 (see Non-patent Document 2), CA125 (see Non-patentDocument 3), and the like.

Meanwhile, it is known that a post-translational modified, inparticular, phosphorylated protein reflects conditions of various organsand tissues. For example, a phosphorylated troponin 1 protein forrecognizing a condition of a muscle tissue that has suffered damage (seePatent Document 1); and phosphorylated urokinase to be used fordetecting various types of carcinoma (see Patent Document 2).

[Non-patent Document 1] Liebman H A, Furie B C, Tong M J, Blanchard R A,Lo K J, Lee S D, Coleman M S, and Furie B., New Engl. J. Med. 310, pp.1427-1431. (1984)

[Non-patent Document 2] Kumagai Y, Chiba J, Sata T, Ohtaki S, andMitamura K. Cancer Res. 52, pp 4987-4994. (1992)

[Non-patent Document 3] Elias J, Kew M C. Int. J. Cancer. 46, pp805-807. (1990)

[Patent Document 1] JP-A-2006-502203, Title of the Invention “ISOLATEDPOST-TRANSLATIONALLY MODIFIED PROTEINS FOR MONITORING AND DIAGNOSINGMUSCLE DAMAGE” [Patent Document 2] JP-B-3129430, Title of the Invention“METHOD FOR DETECTING TUMOR DISEASE” DISCLOSURE OF THE INVENTION Problemto be Solved by the Invention

However, markers disclosed in Non-patent Documents 1 to 3 have not beensufficient in assessment rate of positive results.

For example, the screening rates of AFP and PIVKA-II in hepatocellularcarcinoma assessment are 60% to 70%. Therefore, there is a demand for amarker with additional reliability.

An object of this invention is to provide a protein other than thosedescribed in the above-mentioned Patent Documents 1 and 2, including amethod of assessing hepatocellular carcinoma by using a protein which ispresent in a hepatocellular carcinoma cell in a different phosphorylatedstate from that in a non-hepatic carcinoma cell.

Further, another object of this invention is to provide a hepatocellularcarcinoma protein marker for detecting the hepatocellular carcinoma,including a protein having a different phosphorylated state.

Means to Solve the Problem

According to an aspect of this invention, there is provided ahepatocellular carcinoma protein marker, which includes tumor rejectionantigen gp96 formed of an amino acid represented by SEQ ID NO: 1, inwhich the protein marker is phosphorylated.

According to another aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma which includes using thehepatocellular carcinoma protein marker as above-described.

According to still another aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma as above-described. In themethod, the using the protein marker includes extracting from asurgically sampled biological sample.

According to yet another aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma as above-described. In themethod, the using the protein marker further includes preparing thebiological sample which includes any one kind of a biopsy sample, blood,plasma, serum, and urine.

According to a further aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma as above-described. In themethod, the using the protein marker includes verifying phosphorylationof the protein marker.

According to another aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma as above-described. In themethod, the verification is performed based on measurement of any one ofa serine residue, a threonine residue, and a tyrosine residue of theprotein marker for its phosphorylation.

According to still another aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma as above-described. In themethod, the protein marker is extracted from a surgically sampledbiological sample.

According to a further aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma, which includes measuringphosphorylation of tumor rejection antigen gp96 formed of an amino acidrepresented by SEQ ID NO: 1, and judging the measurements as positivebased on a comparison with a normal value.

According to a still further aspect of this invention, there is provideda method of detecting hepatocellular carcinoma, which includes measuringphosphorylation of tumor rejection antigen gp96 as the hepatocellularcarcinoma protein marker in a biological sample, wherein the measuringis performed by a method selected from the group consisting of an enzymeimmunoassay, a fluorescence-labeled antibody method, a western blotmethod, a radioimmunoassay, an immunoprecipitation method,electrophoresis, liquid chromatography, and mass spectrometry.

According to a yet further aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma as above-described. In themethod, the measuring includes extracting a protein from a surgicallysampled biological sample, subjecting the protein to a multicycle ofelectrophoresis with different dimensions, fixing and staining theprotein on a solid carrier, and comparing an increase or decrease in thetumor rejection antigen gp96 in a protein spot.

According to another aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma as above-described. In themethod, the measuring further includes preparing the biological samplewhich includes any one kind of a biopsy sample, blood, plasma, serum,and urine.

According to still another aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma as above-described. In themethod, the measuring includes judging a presence or absence ofhepatocellular carcinoma based on a comparison of the results obtainedfrom measuring phosphorylation of a digestion product of the tumorrejection antigen gp96 obtained from a surgically-sampled biologicalsample in positive ion mode measurement and in negative ion modemeasurement by using a mass spectrometer.

According to a further aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma as above-described. In themethod, the measuring phosphorylation of the hepatocellular carcinomaprotein marker is performed with respect to any one of a serine residue,a threonine residue, and a tyrosine residue of the protein marker.

According to yet another aspect of this invention, there is provided amethod of detecting hepatocellular carcinoma as above-described. In themethod, the measuring further includes using an antibody that recognizesa phosphorylated site of the tumor rejection antigen gp96.

Effect of the Invention

The measurement of phosphorylation of the tumor rejection antigen gp96formed of the amino acid represented by SEQ ID NO: 1 can be used todetect hepatocellular carcinoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison between gels each stained with a solution forstaining a phosphorylated protein after a protein sample extracted froma non-carcinoma site (a) and a protein sample extracted from a carcinomasite (b), which are obtained from 18 patients, have been developed bytwo-dimensional electrophoresis.

FIG. 2 is a view showing the whole amino acid sequence of tumorrejection antigen gp 96.

FIG. 3 includes charts each showing a MALDI-TOF/MS spectrum of a productobtained by digesting tumor rejection antigen gp96 with trypsin. (a)shows the results in positive ion mode measurement and (b) shows theresults in negative ion mode measurement.

FIG. 4 includes charts each showing a MALDI-TOF/MS spectrum of a productobtained by digesting tumor rejection antigen gp96 with trypsin, andshowing a comparison between the results in positive ion modemeasurement (a) and the results in negative ion mode measurement (b) ina mass range of 2,050 Da to 2,800 Da.

DESCRIPTION OF REFERENCE NUMBERS

-   1 spot of tumor rejection antigen gp96 of sample derived from    non-carcinoma site-   2 spot of tumor rejection antigen gp96 of sample derived from    carcinoma site-   41 peak derived from FQSSHHPTDITSLDQYVER peptide of tumor rejection    antigen gp96 measured in positive ion mode-   42 peak derived from FQSSHHPTDITSLDQYVER peptide of tumor rejection    antigen gp96 measured in negative ion mode-   43 peak derived from FQSSHHPTDITSLDQYVER peptide of phosphorylated    tumor rejection antigen gp96 measured in negative ion mode

BEST MODE FOR CARRYING OUT THIS INVENTION

Hereinafter, this invention is described in more detail.

The inventors of this invention have researched a protein whosephosphorylation is accelerated in hepatocellular carcinoma tissuescompared with non-carcinoma cells. As a result, the inventors have foundthat the phosphorylation amount of a specified protein may be measuredto distinguish hepatocellular carcinoma cells from non-carcinoma cells.Thus, this invention has been completed.

That is, this invention relates to a method of assessing hepatocellularcarcinoma by measuring the variation in phosphorylation of tumorrejection antigen gp96 formed of the amino acid represented by SEQ IDNO: 1 in a biological sample obtained from a subject.

Specifically, this invention provides a method of measuring tumorrejection antigen gp96 formed of the amino acid represented by SEQ IDNO: 1 for its phosphorylation by a method selected from the groupconsisting of an enzyme immunoassay, a fluorescence-labeled antibodymethod, a western blot method, a radioimmunoassay, animmunoprecipitation method, electrophoresis, liquid chromatography, andmass spectrometry.

In this case, the biological sample, such as a biopsy sample, blood,plasma, serum, and urine, can be used in this invention.

Examples of the method of measuring such variation in proteinphosphorylation as described above in such a biological sample include:a method using in combination two-dimensional electrophoresis forseparating a protein from the biological sample, which is a combinationof isoelectric point electrophoresis and SDS polyacrylamide gelelectrophoresis, and a staining method for allowing a phosphorylatedstate of the separated protein to be visualized; a method involvingseparating the biological sample by multidimensional chromatography,which is a combination of various types of chromatography such asion-exchange chromatography, reverse-phase chromatography, and gelfiltration chromatography, and determining the phosphorylation of theseparated protein by mass spectrometry; and a method using a specifiedantibody that recognizes a phosphorylated site of a protein.

Further, the specified antibody according to this invention is anantibody which recognizes a phosphorylated serine residue, aphosphorylated threonine residue, and a phosphorylated tyrosine residue.The protein phosphorylation in a sample may be detected by using thoseantibodies alone or in combination, and employing a known method such asan enzyme immunoassay (ELISA), a western blotting method, aradioimmunoassay, and an immunoprecipitation method.

EXAMPLES

This invention is described in detail by way of examples, but thisinvention is not limited by those examples.

Example 1

In Example 1 of this invention, a two-dimensional electrophoresisanalysis of proteins extracted from carcinoma cells and proteinsextracted from non-carcinoma cells, which are derived fromhepatocellular carcinoma patients, is described.

Carcinoma site tissues and non-carcinoma site tissues, which wereobtained from tissues surgically excised from 18 hepatocellularcarcinoma patients and were diagnosed pathologically, each were crushedin a cell lysis solution (30 mM Tris-Cl (pH 8.5), 7 M urea, 2 Mthiourea, 4% (w/v) CHAPS, 0.5 mM EDTA, PMSF, Aprotinin, and Pepstatin)by using a glass homogenizer, followed by incubation at 37° C. for 1hour. After the sample was centrifuged (13,000 rpm, 20 minutes), thesupernatant was collected. The protein concentration in the supernatantwas measured using a protein assay kit (manufactured by Bio-RadLaboratories, Inc.) by a Bradford method. 25 μg of the protein extractedfrom a carcinoma site and 25 μg of the protein extracted from anon-carcinoma site, which had been sampled from each of the patients,were mixed to prepare a protein sample extracted from a carcinoma site(total: 450 μg) and a protein sample extracted from a non-carcinoma site(total: 450 μg). The protein samples extracted from a carcinoma site andthe protein samples extracted from a non-carcinoma site were subjectedto an isoelectric point electrophoresis at 71,500 Volt/hour by using animmobilized pH gradient gel (Immobiline DryStrip pH 3-10, 24 cm: GEHealthcare) (one-dimensional electrophoresis). The gel after beingsubjected to the one-dimensional electrophoresis was reduced-alkylated,a two-dimensional SDS electrophoresis was performed by using a 12.5%polyacrylamide gel (24 cm×20 cm). The gel subjected to theabove-mentioned separation was stained with a solution for staining aphosphorylated protein (Pro-Q Diamond phosphoprotein gel stain:Invitrogen Corporation). The stained gel was detected for its proteinspots with an image analyzer (Typhoon 9400: GE Healthcare UK Ltd).

A protein spot at which the staining with the solution for staining aphosphorylated protein varied more greatly in the carcinoma sitecompared with the non-carcinoma site was specified. The specifiedprotein spot was cut out from the gel, and converted into a peptide byin-gel tryptic digestion. The in-gel digested peptide was measured withan ion spray mass spectrometer (ESI-MS, LCQ-Deca: ThermoelectronCorporation), and the protein at the spot was identified by a peptidemass fingerprint method (PMF method) using a Mascot software (MATRIXSCIENCE Ltd.).

The results revealed that the staining of the protein spot of the tumorrejection antigen gp96 with the solution for staining a phosphorylatedprotein varied more greatly in the carcinoma site compared with thenon-carcinoma site.

FIG. 1 shows a comparison between gels each stained with the solutionfor staining a phosphorylated protein after the protein sample extractedfrom a carcinoma site and the protein sample extracted from anon-carcinoma site has been developed by two-dimensionalelectrophoresis. The reference numeral 1 in FIG. 1 denotes a spot of thetumor rejection antigen gp96 of a sample derived from the non-carcinomasite, and the reference numeral 2 denotes a spot of the tumor rejectionantigen gp96 of a sample derived from the carcinoma site. With regard tothe respective spots denoted as the reference numerals 1 and 2, thesample derived from the non-carcinoma site (1) and the sample derivedfrom the carcinoma site (2) differ from each other in the staining withthe solution for staining a phosphorylated protein. The spot denoted asthe reference numeral 2 in FIG. 1 was cut out and subjected to in-geltryptic digestion. After that, the protein was identified by the PMFmethod. As a result, as underlined in FIG. 2, a peptide having mass thatcorresponds to a partial peptide of the tumor rejection antigen gp96 wasobserved as a peptide obtained by subjecting the protein spot gel totryptic digestion.

Example 2

In Example 2 of this invention, there is described the verification ofthe tumor rejection antigen gp96 for its phosphorylation by MALDI-TOF/MSmeasurement.

A product obtained by digesting the tumor rejection antigen gp96 withtrypsin was dissolved in 10 μl of an aqueous solution containing 0.1%TFA and 50% methanol to obtain a sample solution. 1 μl of the samplesolution was dropped onto a target plate for a mass spectrometer, anddried at room temperature. To the dried sample spot, dropped were 0.7 μlof a matrix solution (a solution obtained by dissolvingα-cyano-4-hydroxycinnamic acid (α-CHCA) in a solution containing 0.1%trifluoroacetic acid (TFA), 70% methanol, and 4 mM mono ammoniumphosphate so that the concentration would be a saturated concentration),followed by drying at room temperature. The target plate was measuredwith a time-of-flight mass spectrometer (MALDI-TOF/MS, Voyager DE STR:Applied Biosystems, Inc.) in a linear mode, and the ion polarity to bemeasured was measured in two ways, i.e., a positive ion mode and anegative ion mode. The mass in the spectrum was calibrated by anexternal standard method using a standard peptide.

FIG. 3 includes charts showing a comparison between the results inpositive ion mode measurement and the results in negative ion modemeasurement in a mass range of 699 Da to 3,001 Da. The negative ion modecan measure a mass spectrum of a phosphorylated peptide difficult to bemeasured in the positive ion mode.

FIG. 4 includes charts showing a comparison between mass spectra in amass range of 2,050 Da to 2,800 Da. In FIG. 4, the reference numeral 41denotes a peak derived from the FQSSHHPTDITSLDQYVER peptide of the tumorrejection antigen gp96 measured in the positive ion mode. Further, thereference numeral 42 denotes a peak derived from the FQSSHHPTDITSLDQYVERpeptide of the tumor rejection antigen gp96 measured in the negative ionmode. Still further, 43 denotes a peak derived from theFQSSHHPTDITSLDQYVER peptide of the phosphorylated tumor rejectionantigen gp96 measured in the negative ion mode. As clear from FIG. 4,there is a peak observed only in the negative ion mode measurement (43in FIG. 4) by a mass difference of +80 Da from a peak commonly observedin both of the positive ion mode measurement and the negative ion modemeasurement (41 in FIG. 4 and 42 in FIG. 4). The mass of the peakcommonly observed in both of the positive ion mode measurement and thenegative ion mode measurement (41 in FIG. 4 and 42 in FIG. 4) coincideswith the mass of the peptide of 512th to 530th residues(FQSSHHPTDITSLDQYVER) in the tumor rejection antigen gp96, and it isconceivable that any one of a serine residue (S), a threonine residue(T), and a tyrosine residue (Y) in FQSSHHPTDITSLDQYVER has beensubjected to phosphorylation.

Accordingly, the measurement of the tumor rejection antigen gp96 for itsphosphorylation is useful for diagnosis of hepatocellular carcinoma.

INDUSTRIAL APPLICABILITY

This invention can provide the hepatocellular carcinoma protein markerand the method of detecting hepatocellular carcinoma cells using thehepatocellular carcinoma protein marker.

Note that the application of this invention insists advantage thereofbased on the priority of Japanese Patent Application No. 2007-162857filed on 20 Jun., 2007, and the disclosure of the filed application istaken into the whole of this application.

1. A method of detecting hepatocellular carcinoma, comprising: using anisolated protein comprising an amino acid sequence represented by SEQ IDNO:
 1. 2. The method of detecting hepatocellular carcinoma according toclaim 1, wherein the using of the isolated protein includes extractingfrom a surgically-sampled biological sample.
 3. The method of detectinghepatocellular carcinoma according to claim 2, wherein the using of theisolated protein further comprises preparing the biological samplecomprising any one kind of a biopsy sample, blood, plasma, serum, andurine.
 4. The method of detecting hepatocellular carcinoma according toclaim 1, wherein the using of the isolated protein includes verifyingphosphorylation of the isolated protein.
 5. The method of detectinghepatocellular carcinoma according to claim 4, wherein the using of theisolated protein further includes extracting from a surgically sampledbiological sample.
 6. The method of detecting hepatocellular carcinomaaccording to claim 5, wherein the using of the protein marker furthercomprises preparing the biological sample comprising any one kind of abiopsy sample, blood, plasma, serum, and urine.
 7. The method ofdetecting hepatocellular carcinoma according to claim 4, wherein theverifying phosphorylation of the isolated protein is performed based onmeasurement of any one of a serine residue, a threonine residue, and atyrosine residue of the isolated protein for its phosphorylation.
 8. Themethod of detecting hepatocellular carcinoma according to claim 7,wherein the using of the isolated protein comprises extracting theisolated protein from a surgically sampled biological sample.
 9. Themethod of detecting hepatocellular carcinoma according to claim 8,wherein the using of the isolated protein further comprises preparingthe biological sample comprising any one kind of a biopsy sample, blood,plasma, serum, and urine.